Calcium-binding affinity and calcium-enhanced activity of Clostridium thermocellum endoglucanase D.
Clostridium thermocellum endoglucanase D (EC 3.2.1.4: EGD), which is encoded by the celD gene, was found to bind Ca2+ with an association constant of 2.03 x 10(6) M-1. Ca2+ stimulated the activity of EGD towards swollen Avicel by 2-fold. In the presence of Ca2+, the Kd of the enzyme towards p-nitrophenyl-beta-D-cellobioside and carboxymethylcellulose was decreased by 4-fold. Furthermore, Ca2+ increased the half-life of the enzyme at 75 degrees C from 13 to 47 min. Since the 3' sequence of celD encodes a duplicated region sharing similarities with the Ca2+-binding site of several Ca2+-binding proteins, a deleted clone was constructed and used to purify a truncated form of the enzyme which no longer contained the duplicated region. The truncated enzyme was very similar to EGD expressed from the intact gene with respect to activity, Ca2(+)-binding kinetics and Ca2+ effects on substrate binding and thermostability. Thus the latter parameters do not appear to be mediated through the duplicated conserved region.
Chauvaux
S
S
Unité de Physiologie Cellulaire and URA 1300 CNRS, Départment des Biotechnologies, Institut Pasteur, Paris, France.
Beguin
P
P
Aubert
J P
JP
Bhat
K M
KM
Gow
L A
LA
Wood
T M
TM
Bairoch
A
A
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism*; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010957:Plasmids; D012689:Sequence Homology, Nucleic Acid
D002480:Cellulase; D002118:Calcium